When treating many pathologies, which lead to the degeneration of specific organs, the only possible therapeutic alternative is often represented by the organ transplantation. Where possible, however, it is associated to a high grade of risk for the patient, since complications are likely to occur, such as the transmission of pathologies from donor to recipient or the rejection of the organ itself, along with the chronic shortage of available organs. Moreover, such an approach cannot be applied to all the body zones susceptible to degenerative pathologies. Actually, at present, transplant therapies may not be applied to many degenerative pathologies, such as, in some cases, those related to some zones of the connective tissues, bone tissue, dermis and epidermis, besides the central nervous system and sometimes also the myocardium.
Since most cells of the central nervous system, for example neurons, do not proliferate nor renew, attention has been addressed to therapies based on replacement or regeneration of the damaged tissue, using isolated and re-grafted cells. In the recent history, the neural tissue engineering has mainly focused on Parkinson's disease [See, for example: Tabar et al., Nature Medicine (2008), doi:10,1038/nm1732] and on lesions in the spinal marrow [See, for example: Perale et al. Journal of Applied Biomaterials and Biomechanics (2008) vol. 6 pp. 1-8; Huang et al. Journal of Neurosurgery (2008) vol. 108 pp. 343-347; Nomura et al Journal of Neurotrauma (2006) vol. 23 pp. 496-507; Iwata et al. Tissue Engineering (2006) vol. 12 pp. 101-110], the relationships cause-effect involved in such diseases being well-known and effective therapies for treating the same being not available yet. In such a field, first experiments have been carried out, by directly transplanting or injecting aqueous suspensions of individual cells or cell populations.
The myocardial infarction pathologies represent a further well-known, representative case. Indeed, these are a common cause of death and lower quality of life all around the world. Following a heart attack, the myocardium suffers a more or less extended damage, which leads to lose the heart functionality and necrotic and non-functional tissues to be formed. When death is prevented, the chronic heart failure, at different levels, is a sure perspective. At the present state, for more serious stages of this pathology, effective pharmacological treatments are not available yet, and therefore, the transplantation is often the last, but unfortunately not always feasible, therapeutic opportunity. For less serious cases, the available treatments allow only to keep the clinical status quo. Therapies, which lead to significant improvement and recover for the myocardium, in fact, are not available [See, the monograph J. O. Mudd, D. A. Kass, Nature, volume 451: pp. 919-928, 2008]. The likelihood to inject specific healthy cells in the infarcted zones in order to cause the regeneration is arising as a possibly more successful way [as it is evident, for example, in the following monographs: M. N. Giraud et al., Tissue Eng 13(8): 1825-1836; 2008. V. F. M. Segers, R. T. Lee, Nature 451: 937-942; 2008. S. J. Dimmeler et al., Arterioscl. Thromb. Vasc. Biol. 28: 208-216, 2008].
The lower level of cell specialization in tissues with a strong connective component represents, perhaps, a better condition from several points of view, as some opportunities exist, to make bi- and tri-dimensional artificial structures capable of hosting and carrying specific cells for the functional recovery of damaged structures. These artificial devices, also known as scaffolds, represent one of the pillars of tissue engineering, but, of course, they are not free of problems [As an example, see the monograph C. H. Evans et al., Tissue Engineering, Volume 13(8), pp. 1987-1993, 2007]. In this field, a well known kind of damages is linked to those related to the cartilages, mostly the articular cartilages. The cartilaginous tissue is a particular kind of connective tissue, characterized by remarkable resistance and flexibility properties, which plays a role of structural support within the organism. It is a particular kind of connective tissue and, as such, it is composed of cells dispersed in much gelatinous extracellular matrix, rich in fibres (responsible for elasticity) and an amorphous substance originated from proteins. Articular cartilages are those, which cover the joint surfaces and those most known for being susceptible to traumas are those related to the knee. Of course, they are not lethal pathologies, but they are annoying and can lower the quality of life and be disabling. The surgical techniques developed aim to substantially reduce the defect, by eliminating the injured zone, but this exposes the joint to more friction and wear over the time. There are no substantially effective pharmacological treatments. Therefore, the regenerative medicine is deemed as the frontier for treating these pathologies, mostly those originating from traumas. Carrying chondrocytes and making them remain alive in situ is the basic setting for therapies under development, mainly based on two similar approaches: a) carrying the cells by using planar solid matrixes in the form of the defect to be integrated, b) carrying the cells through gels, which are injected until the defect to be integrated is filled. In both cases, the solutions tend to use biodegradable materials, in order to promote the integration [as shown, for instance, in the following monographs: Jorgensen C et al., Best Practice & Research In Clinical Rheumatology 22(2), pp. 269-284, 2008. Grayson W L et al., Trends In Biotechnology, (26)4, pp. 181-189, 2008].
Likewise, the degenerative pathologies regarding the bone tissues can result in a particularly severe structural fragility and the main therapies are pharmacological, which, however, do not always guarantee efficacy and are not devoid of side effects. However, also the tissue engineering sector is bringing new perspectives to the bone regeneration [as it can be seen from the note reported in the following work: Waese E Y L, Kandel R R, Stanford W L, Skeletal Radiology 37(7), pp. 601-608, 2008]. In this field, a fundamental role is played by the mechanical properties of the materials used for making the scaffolds, which are the most usable devices [as reported in several monographic works, among which the following can be mentioned: Stevens M M, Materials Today 11(5), pp. 18-25, 2008. Barrere F et al., Materials Science & Engineering R-Reports, 59(1-6), pp. 38-71, 2008]. For sake of completeness, it is to be reminded that in the bone, tendon and cartilage regeneration field, several investigations exist, which provide the use of highly-porous scaffolds imbibed with hydrogel-based matrices, containing, in turn, the cells [See, to this end, for example, the wide bibliography cited in the U.S. Pat. No. 6,171,610].
Generally, briefly, the scientific literature substantially identifies four possible paths to regenerate tissues:
a) stimulating independent endogenous regeneration mechanisms—an approach founded on bases, which are not solid yet, and with procedures, not well-identified yet, as a whole not really promising at the moment;
b) transplanting isolated cells directly in the injured site—an empirical approach, based on a good functional principle, but that brings along a number of drawbacks and problems, such as the high cell loss and the resulting low integration with the adjacent tissue;
c) transplanting tissue generated in vitro (also called graft)—an empirical approach based on a good tissue principle, but with remarkable complexities, since the graft needs to integrate with the adjacent tissues, at its best, be capable of withstanding the possible mechanical stress it undergoes once in vivo and, possibly, have a suitable bioabsorbability with the target tissue;
d) using advanced micro- and nano-structured materials, which are capable of hosting specific cell populations providing a compatible and favourable environment, which, subsequently, can be used as a carrier for the implant—futuristic approach, based on rational principles, but that still needs a full comprehension of the phenomena involved.
Whatever approach is followed, the procedures presently available often allow only a partial, if any, reconstruction to be achieved. This limited success depends on several reasons, and, mainly, they have to be found in the cellular aspects and in the lack of a suitable physical support for the thus transplanted, or injected, cells, during the time between the implant and the integration with the adjacent tissues. Moreover, when a suitable nutrient-permeable support is missing, cells cannot obtain the necessary nutrients to survive and eliminate the catabolytes.
The above reasons are leading the research to be addressed towards the development of support matrixes, which allow to carry cells, allowing and/or increasing the same to survive and integrate with the target tissue, in order to give rise to a new functional tissue, histologically integrated with the adjacent structures.
In view of the foregoing, besides the cells, an essential element of tissue engineering is therefore represented by the support and carrier materials used, among these, hydrogels play a key role, since they are useful as support and carrier systems for cells and cell populations.
However, even though the literature about this topic is quite comprehensive, the specifically suitable hydrogels for hosting cells, particularly of the central nervous system, are a minority. To this end, see what is reported in the following literature: Huang et al. Journal of Neurosurgery (2008) vol. 108 pp. 343-347; Crompton et al. Biomaterials (2007) vol. 28 pp. 441-449; Frampton et al. Journal of Neural Engineering (2007) vol. 4 pp. 399-409; Hind et al. Journal of Biomaterial Science Polymer Edition (2007) vol. 18 pp. 1223-1244; Mahoney et al. Biomaterials (2006) vol. 27 pp. 2265-2274; Prang et al. Biomaterials (2006) vol. 27 pp. 3560-3569; Luo et al. Nature Materials (2004) vol. 3 249-253; Vacanti et al. Transplantation Proceedings (2001) vol. 33 pp. 592-598; Woerly, EP0929323). This is mainly due to the difficulties in making hydrogels, which allow the cells, in particular the central nervous system cells, to survive and grow therein, by providing them both support and a suitable viable space.
In some cases, the mechanical functionality requirements make the use of scaffolds indispensable, but the target tissues very often require to be inundated by a cell flow, which is stable and allows the reintegration thereof, and this can be achieved by using proper hydrogels. As already stated, in some cases, mixed solutions may be used, wherein the solid scaffolds are imbibed with hydrogels containing or supporting the cells.
Hydrogels are tri-dimensional polymeric structures, capable of swelling by retaining water, or other liquids, therein. The polymeric chains are linked to each other through intermolecular bonds, which can have a different origin. Hydrogels can be produced in a number of ways, one of which consists in reacting one or more monomers or due to the association of hydrogen bridges or strong van der Waals interactions among polymeric chains. According to the components used, the hydrogels can be biodegradable or biostable, biocompatible or cytotoxic. A lot of biocompatible, biodegradable or not, hydrogels are gaining interest from the technical-scientific world, due to their inherent characteristics, which make them particularly suitable and ideal for many applications in the bio-medical field.
From a strictly chemical point of view, hydrogels can be classified in several ways, according to their preparation method, the distributed ion charge or their physical structure. In relation to the preparation, hydrogels can be classified as homopolymeric, co-polymeric, multi-polymeric or interpenetrating polymeric hydrogels. The firsts are made by polymeric networks, bound to each other by a monomer unit, which has to be hydrophilic. The seconds are made of two co-monomers, at least one of which is to be hydrophilic. The multi-polymeric ones are obtained starting from three or four co-monomers interacting with each other, at least one being hydrophilic. Finally, the interpenetrating polymeric ones are obtained through swelling a first network, around which, subsequently, a tri-dimensional structure is formed.
Therefore the chemical and physical properties of hydrogels make them particularly suitable for being used in the biomedical and pharmaceutical fields. In particular, their biocompatibility is the first fundamental requirement for such applications.
Their hydrophilicity can guarantee optimal features in terms of gas- and other substances-permeability, allowing also the controlled release of active substances and supporting the presence of cell populations inside or on their surface. One of the first applications developed, still widely spread today, is linked to manufacturing contact lenses where hydrogels are used due to their good mechanical stability, the favourable refractive index and their high oxygen permeability. Other applications, such as those described in the present application, include using hydrogels as bio-adhesive artificial materials, artificial membranes, articular cartilages, artificial skin, materials for the maxillo-facial and vocal cord reconstruction.
It is to be highlighted that hydrogels find a wide use also as in vitro cell growth support, in pharmacological screening applications, drug-development assay, pharmacological efficacy or toxicity or other electro-physiological or bio-mechanical features. Some of these assays are carried out through devices, also known as bioreactors and “biochips”.
For example, such supports are usually configured as thin films, on which neurons are laid and grown. Therefore, they are bi-dimensional structures on which neurons are laid on one of the surfaces and not placed therein. Among the other hydrogels, devised and developed for these uses, the most used are Matrigel™ (Beckton Dickinson, USA) and fibrin or collagen gel (Viscofan S/A, Spain).
It is to be reminded that the recent advances in electronics have made available for neuroscientists several technological solutions, which allow the communication between specific electronic devices and living neuronal cells. Measuring devices are, in fact, available on the market, specifically made for monitoring the cell activity of individual neurons or populations of neurons, during several kinds of testing, ranging from monitoring the signal conductivity to pharmacological efficacy. In such electronic systems, the contact between neurons and an electronic substrate is often mediated by a thin (a few microns) film of polymeric hydrogel, suitably designed and made in order to allow the passage of both electrical and chemical signals.
The technical-scientific literature available, regarding the materials, including hydrogels, and their use in the regenerative field, is very wide, in the nervous regeneration field, it is to be considered the recent review carried out by Little et al. Chemical Reviews (2008) vol. 108 pp. 1787-1796, and in this wide survey, hydrogels are, per se, known and they are often described as means for controlled drug-delivery and supports for tissue engineering [as it can be inferred from the monographic reviews cited below as a way of example: J. D. Kretlow et al., Advanced Drug Delivery Reviews 59 pp. 263-273, 2007. L. Yu et al., Tutorial Review on Chemical Society Reviews, 2008].
The preparation of several anionic gels for the controlled drug delivery was described in 1986 by Hsu et al. in Pharmaceutical Research (1996) vol. 13 pp. 1865-1870. In particular, the preparation of a two-component hydrophilic gel made of an agarose and carbomer formulation. This particular gel is prepared by mixing an aqueous carbomer dispersion with a pre-heated agarose dispersion at a certain temperature.
The European patent EP0929323 describes a biostable (and therefore biodegradable) polymeric hydrogel for therapeutic use, consisting in a copolymer of (a) an N-substituted methacrylamide or acrylamide (b) a cross-linking agent and (c) co-polymerisable material, which can be used for reconstructing the damaged cerebral or spinal tissue.
The European patent EP1206254 describes a tri-dimensional matrix based on re-absorbable fibers, loaded with specific drugs, wherein cells are grown in vitro, and are subsequently implanted in vivo.
The patent application US2007010831 describes an implant for repairing damaged nerves, comprising an external biocompatible perforated conduit and an inner hydrogel matrix made of agar, agarose, xanthan gum, carbopol, alginate salts, polyvinylpyrrolidone, polyethylene glycole, chitosane, cellulose, acrylics and polyglycolic polymers and other natural or synthetic polymers.
U.S. Pat. No. 6,171,610 is also mentioned, where formulations are described, obtained by mixing biocompatible substances, and their extended applications to all the possible cell populations and in particular to the central nervous system tissue.